We have investigated the effects of supraphysiological concentrations of catecholamines on glucocorticoid secretion in vitro. These effects were analyzed in adrenocortical cells shown to be present in chromaffin cell cultures as well as in cortical cells cocultured with transfected chromaffin cells that overproduce catecholamines. Cortisol release from residual cortical cells in chromaffin cell cultures was found to be 2.5 times higher than from isolated adrenocortical cells. Removal of the adrenocortical cells from the chromaffin cells resulted in an almost complete cessation of cortisol secretion. Catecholamine overproduction was achieved by transfecting chromaffin cells with the blank retroviral vector pSAM-EN. Coculture of adrenocortical cells with these transfected chromaffin cells further enhanced the stimulating effect of chromaffin cells on cortisol 2.3-fold compared to normal cocultures. In conclusion, cortical cells in chromaffin cell cultures secrete significant amounts of cortisol, which should be considered when evaluating the endocrine function of these cell cultures and which can be abolished by purification. The hormonal activity of adrenocortical cells is highly increased in an environment of catecholamine overproduction, which is of both basic and clinical importance.